KC-1293

Ba/F3-FIP1L1-PDGFRa-D842V-Cell-Line

21873

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Background of Ba/F3-FIP1L1-PDGFRa-D842V-Cell-Line

Platelet-derived growth factor receptor A, abbreviated as PDGFRA or PDGFRα, is a cell-surface receptor tyrosine kinase, and activated by binding of its specific ligand, platelet-derived growth factors (PDGFs), play an important role in cell growth and differentiation. The overactivation of PDGFRA due to mutation and/or chromosome fusion are associated with hematological malignancy, and gastrointestinal stromal tumors (GISTs). The identification of PDGFRA as a driver gene has led to the development of anticancer therapeutics agents, including Avapritinib and Olaratumab. Ba/F3 cell, a murine interleukin-3 dependent pro-B cell line, is a popular system for exploring both kinases and their inhibitors, because some protein kinases can render the Ba/F3 cells to be depended on the activation of the kinases instead of IL-3 supplement, while their inhibitors can antagonize the kinase-dependent growth effects.

Specifications

Catalog Number	KC-1293
Cell Line Name	Ba/F3-FIP1L1-PDGFRa-D842V-Cell-Line
Description	Stable Ba/F3 clone expressing exogenous FIP1L1-PDGFRA fusion protein bearing D842V mutation in PDGFRA part.
Quantity	One vial of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Mostly single, round (some polymorph) cells in suspension
Subculture	Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Ba/F3 FIP1L1-PDGFRA-D842V cell line was generated using retrovirus vector expressing human FIP1L1-PDGFRA-D842V sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 1µg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:10 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Montano-Almendras, C P, A Essaghir, H Schoemans, I Varis, L A Noel, A I Velghe, D Latinne, L Knoops, and J B Demoulin. 2012. ¡ùETV6-PDGFRB and FIP1L1-PDGFRA Stimulate Human Hematopoietic Progenitor Cell Proliferation and Differentiation Into Eosinophils: the Role of Nuclear Factor- B.¡ì Haematologica 97 (7): 1064Ã¿72.
Nielsen, B S, R R Malinda, F M Schmid, S F Pedersen, S T Christensen, and L B Pedersen. 2015. ¡ùPDGFR and Oncogenic Mutant PDGFR D842V Promote Disassembly of Primary Cilia Through a PLC - and AURKA-Dependent Mechanism.¡ì Journal of Cell Science 128 (19): 3543Ã¿49. doi:10.1242/jcs.173559.
Bahlawane, Christelle, Ren¹È Eulenfeld, Monique Y Wiesinger, Jiali Wang, Arnaud Muller, Andreas Girod, Petr V Nazarov, et al. 2015. ¡ùConstitutive Activation of Oncogenic PDGFRÏ«-Mutant Proteins Occurring in GIST Patients Induces Receptor Mislocalisation and Alters PDGFRÏ« Signalling Characteristics.¡ì Cell Communication and Signaling 13 (1): 49Ã¿21. doi:10.1186/s12964-015-0096-8.
Montano-Almendras, C P, A Essaghir, H Schoemans, I Varis, L A Noel, A I Velghe, D Latinne, L Knoops, and J B Demoulin. 2012. ¡ùETV6-PDGFRB and FIP1L1-PDGFRA Stimulate Human Hematopoietic Progenitor Cell Proliferation and Differentiation into Eosinophils: the Role of Nuclear Factor- B.¡ì Haematologica 97 (7): 1064Ã¿72. doi:10.3324/haematol.2011.047530.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.